Netrin-1 improves post-injury cardiac function in vivo via DCC/NO-dependent preservation of mitochondrial integrity, while attenuating autophagy

Abstract

Reperfusion injury of the heart is a severe complication of angioplasty treatment of acute myocardial ischemia, for which no therapeutics are currently available. The present study aimed to identify whether and how a novel protein, netrin-1, induces cardioprotection in vivo during ischemia/reperfusion (I/R) injury. Wild type (WT) C57BL6/J mice were subjected to a 30 min coronary occlusion followed by a 24h reperfusion with vehicle (normal saline), netrin-1, UO126 (MEK1/2 inhibitor), PTIO (nitric oxide/NO scavenger), netrin-1/UO126 or netrin-1/PTIO intraventricularly. Some were injected of netrin-1 via tail vein. Netrin-1 at 5μg/kg induced a substantial reduction in infarct size (19.7 ± 5.0% from 41.3 ± 1.8% in the controls), and markedly improved cardiac function as measured by ejection fraction and fractional shortening from echocardiography. Experiments with mice deficient in netrin-1 receptor DCC (deleted in colorectal cancer, DCC+/-), or reperfusion with netrin-1/UO126 or netrin-1/PTIO, attenuated the protective effects of netrin-1, implicating intermediate roles of DCC, ERK1/2 and NO. Netrin-1 induced phosphorylation of ERK1/2 and eNOS was abolished in DCC+/-mice. Electron spin resonance (ESR) determination of NO production from isolated left ventricles demonstrated that netrin-1 improves NO bioavailability, which was attenuated by UO126 or in DCC+/-mice, suggesting upstream roles of DCC and ERK1/2 in NO production. Netrin-1 further reduced mitochondrial swelling and mitochondrial superoxide production, which was absent when co-treated with PTIO or UO126, or in DCC+/-mice, indicating critical roles of DCC, ERK1/2 and NO in preserving mitochondrial integrity. In a permanent coronary ligation model of myocardial infarction (MI) to assess post-MI remodeling, netrin-1 abolished the marked increase in autophagy. In summary, our data demonstrate robust cardioprotective effect of netrin-1 in vivo, as shown by reduced infarct size and improved cardiac function. Mechanistically, this protection is mediated by netrin-1 receptor DCC, and NO dependent preservation of mitochondria. This work clearly establishes a therapeutic potential of netrin-1 for acute treatment of MI, perhaps also for chronic post-MI remodeling. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases.

Keywords: Autophagy; Ischemia reperfusion (I/R) injury; Myocardial infarction (MI); NADPH oxidase isoforms 4 (NOX4); Netrin-1; Nitric oxide (NO).

Figure 1. Netrin-1 attenuates I/R induced myocardial infarct in vivo

Myocardial infarction was induced by a 30 min of left coronary artery (LCA) ligation followed by a 24 hr of reperfusion in wild type C57BL6 mice in vivo. The mice were treated at the onset of reperfusion with either netrin-1 (1–50 μg/kg) or vehicle (normal saline). Evans blue was used to visualize the non-ischemic area. A) Representative TTC-stained heart slices from I/R (vehicle) group, and I/R+Netrin-1 (1–50 μg/kg) groups. The white area indicates infarct zone, while the blue area indicates non infarcted area. The red and white areas represent area at risk. B) Infarct size analyzed by: percentage of area at risk divided by left ventricular (AAR/LV), infarct size divided by area at risk (Inf/AAR), and infarct size divided by left ventricular (Inf/LV). The results are presented as Mean±SEM. The number of animal per group was seven. ***p<0.001 vs. Control I/R (vehicle).

Figure 1. Netrin-1 attenuates I/R induced myocardial infarct in vivo

Myocardial infarction was induced by a 30 min of left coronary artery (LCA) ligation followed by a 24 hr of reperfusion in wild type C57BL6 mice in vivo. The mice were treated at the onset of reperfusion with either netrin-1 (1–50 μg/kg) or vehicle (normal saline). Evans blue was used to visualize the non-ischemic area. A) Representative TTC-stained heart slices from I/R (vehicle) group, and I/R+Netrin-1 (1–50 μg/kg) groups. The white area indicates infarct zone, while the blue area indicates non infarcted area. The red and white areas represent area at risk. B) Infarct size analyzed by: percentage of area at risk divided by left ventricular (AAR/LV), infarct size divided by area at risk (Inf/AAR), and infarct size divided by left ventricular (Inf/LV). The results are presented as Mean±SEM. The number of animal per group was seven. ***p<0.001 vs. Control I/R (vehicle).

Figure 2. Intraventricular and Intravenous Delivery of Netrin-1 Improves Cardiac Function after I/R Injury in vivo

Echocardiography was performed on wild type C57BL6 mice reperfused for 24 hrs or 72 hrs after a 30 min left coronary artery (LCA) ligation. Left ventricular internal diastole diameter (LVID:D), left ventricular internal systolic diameter (LVID:S), ejection fraction and fractional shortening were measured. A) Representative echocardiography data from mice with netrin-1 (5 μg/kg) injected through the LV at the onset of reperfusion. B) Grouped data for left ventricular internal dimension (LVID) during systole (*p<0.05, **p<0.01 vs. I/R(vehicle)-1 day, ##p<0.01, ###p<0.001 vs. I/R(vehicle)-3 day) and diastole (*p<0.05 vs. I/R(vehicle)-1, # p<0.05 vs. I/R(vehicle)-3 day) for mice with netrin-1 injected through LV, n=3–7/group. C) Grouped ejection fraction and fractional shortening data for mice with netrin-1 injected through LV. *** p<0.001 vs. I/R (vehicle)-1 day, ###p<0.001 vs. I/R (vehicle)-3 day, n=3–7/group. D) Representative echocardiography data from mice with netrin-1 (5 μg/kg) injected through tail vein. E) Grouped LVID during systole (**p<0.01 vs. I/R(vehicle)-1 day, ##p<0.01, ###p<0.001 vs. I/R(vehicle)-3 day) and diastole (*p<0.05, **p<0.01 vs. I/R(vehicle)-1 day, #p<0.05, ##p<0.01 vs. I/R(vehicle)-3 day) from mice with netrin-1 (5 μg/kg) injected through tail vein, n=3–9/group. F) Grouped ejection fraction (*p<0.05, **p<0.01, ***p<0.001 vs. I/R (vehicle) -1 day, ##p<0.01, ###p<0.001 vs. I/R (vehicle) -3 day) and fractional shortening (* p<0.05, ***p<0.001 vs. I/R (vehicle) -1 day, #p<0.05, ###p<0.001 vs. I/R (vehicle) -3 day) from mice with netrin-1 (5 μg/kg) injected through tail vein, n=3–9/group. Data are presented as Mean ± SEM.

Figure 2. Intraventricular and Intravenous Delivery of Netrin-1 Improves Cardiac Function after I/R Injury in vivo

Echocardiography was performed on wild type C57BL6 mice reperfused for 24 hrs or 72 hrs after a 30 min left coronary artery (LCA) ligation. Left ventricular internal diastole diameter (LVID:D), left ventricular internal systolic diameter (LVID:S), ejection fraction and fractional shortening were measured. A) Representative echocardiography data from mice with netrin-1 (5 μg/kg) injected through the LV at the onset of reperfusion. B) Grouped data for left ventricular internal dimension (LVID) during systole (*p<0.05, **p<0.01 vs. I/R(vehicle)-1 day, ##p<0.01, ###p<0.001 vs. I/R(vehicle)-3 day) and diastole (*p<0.05 vs. I/R(vehicle)-1, # p<0.05 vs. I/R(vehicle)-3 day) for mice with netrin-1 injected through LV, n=3–7/group. C) Grouped ejection fraction and fractional shortening data for mice with netrin-1 injected through LV. *** p<0.001 vs. I/R (vehicle)-1 day, ###p<0.001 vs. I/R (vehicle)-3 day, n=3–7/group. D) Representative echocardiography data from mice with netrin-1 (5 μg/kg) injected through tail vein. E) Grouped LVID during systole (**p<0.01 vs. I/R(vehicle)-1 day, ##p<0.01, ###p<0.001 vs. I/R(vehicle)-3 day) and diastole (*p<0.05, **p<0.01 vs. I/R(vehicle)-1 day, #p<0.05, ##p<0.01 vs. I/R(vehicle)-3 day) from mice with netrin-1 (5 μg/kg) injected through tail vein, n=3–9/group. F) Grouped ejection fraction (*p<0.05, **p<0.01, ***p<0.001 vs. I/R (vehicle) -1 day, ##p<0.01, ###p<0.001 vs. I/R (vehicle) -3 day) and fractional shortening (* p<0.05, ***p<0.001 vs. I/R (vehicle) -1 day, #p<0.05, ###p<0.001 vs. I/R (vehicle) -3 day) from mice with netrin-1 (5 μg/kg) injected through tail vein, n=3–9/group. Data are presented as Mean ± SEM.

Figure 2. Intraventricular and Intravenous Delivery of Netrin-1 Improves Cardiac Function after I/R Injury in vivo

Echocardiography was performed on wild type C57BL6 mice reperfused for 24 hrs or 72 hrs after a 30 min left coronary artery (LCA) ligation. Left ventricular internal diastole diameter (LVID:D), left ventricular internal systolic diameter (LVID:S), ejection fraction and fractional shortening were measured. A) Representative echocardiography data from mice with netrin-1 (5 μg/kg) injected through the LV at the onset of reperfusion. B) Grouped data for left ventricular internal dimension (LVID) during systole (*p<0.05, **p<0.01 vs. I/R(vehicle)-1 day, ##p<0.01, ###p<0.001 vs. I/R(vehicle)-3 day) and diastole (*p<0.05 vs. I/R(vehicle)-1, # p<0.05 vs. I/R(vehicle)-3 day) for mice with netrin-1 injected through LV, n=3–7/group. C) Grouped ejection fraction and fractional shortening data for mice with netrin-1 injected through LV. *** p<0.001 vs. I/R (vehicle)-1 day, ###p<0.001 vs. I/R (vehicle)-3 day, n=3–7/group. D) Representative echocardiography data from mice with netrin-1 (5 μg/kg) injected through tail vein. E) Grouped LVID during systole (**p<0.01 vs. I/R(vehicle)-1 day, ##p<0.01, ###p<0.001 vs. I/R(vehicle)-3 day) and diastole (*p<0.05, **p<0.01 vs. I/R(vehicle)-1 day, #p<0.05, ##p<0.01 vs. I/R(vehicle)-3 day) from mice with netrin-1 (5 μg/kg) injected through tail vein, n=3–9/group. F) Grouped ejection fraction (*p<0.05, **p<0.01, ***p<0.001 vs. I/R (vehicle) -1 day, ##p<0.01, ###p<0.001 vs. I/R (vehicle) -3 day) and fractional shortening (* p<0.05, ***p<0.001 vs. I/R (vehicle) -1 day, #p<0.05, ###p<0.001 vs. I/R (vehicle) -3 day) from mice with netrin-1 (5 μg/kg) injected through tail vein, n=3–9/group. Data are presented as Mean ± SEM.

Figure 2. Intraventricular and Intravenous Delivery of Netrin-1 Improves Cardiac Function after I/R Injury in vivo

Echocardiography was performed on wild type C57BL6 mice reperfused for 24 hrs or 72 hrs after a 30 min left coronary artery (LCA) ligation. Left ventricular internal diastole diameter (LVID:D), left ventricular internal systolic diameter (LVID:S), ejection fraction and fractional shortening were measured. A) Representative echocardiography data from mice with netrin-1 (5 μg/kg) injected through the LV at the onset of reperfusion. B) Grouped data for left ventricular internal dimension (LVID) during systole (*p<0.05, **p<0.01 vs. I/R(vehicle)-1 day, ##p<0.01, ###p<0.001 vs. I/R(vehicle)-3 day) and diastole (*p<0.05 vs. I/R(vehicle)-1, # p<0.05 vs. I/R(vehicle)-3 day) for mice with netrin-1 injected through LV, n=3–7/group. C) Grouped ejection fraction and fractional shortening data for mice with netrin-1 injected through LV. *** p<0.001 vs. I/R (vehicle)-1 day, ###p<0.001 vs. I/R (vehicle)-3 day, n=3–7/group. D) Representative echocardiography data from mice with netrin-1 (5 μg/kg) injected through tail vein. E) Grouped LVID during systole (**p<0.01 vs. I/R(vehicle)-1 day, ##p<0.01, ###p<0.001 vs. I/R(vehicle)-3 day) and diastole (*p<0.05, **p<0.01 vs. I/R(vehicle)-1 day, #p<0.05, ##p<0.01 vs. I/R(vehicle)-3 day) from mice with netrin-1 (5 μg/kg) injected through tail vein, n=3–9/group. F) Grouped ejection fraction (*p<0.05, **p<0.01, ***p<0.001 vs. I/R (vehicle) -1 day, ##p<0.01, ###p<0.001 vs. I/R (vehicle) -3 day) and fractional shortening (* p<0.05, ***p<0.001 vs. I/R (vehicle) -1 day, #p<0.05, ###p<0.001 vs. I/R (vehicle) -3 day) from mice with netrin-1 (5 μg/kg) injected through tail vein, n=3–9/group. Data are presented as Mean ± SEM.

Figure 2. Intraventricular and Intravenous Delivery of Netrin-1 Improves Cardiac Function after I/R Injury in vivo

Echocardiography was performed on wild type C57BL6 mice reperfused for 24 hrs or 72 hrs after a 30 min left coronary artery (LCA) ligation. Left ventricular internal diastole diameter (LVID:D), left ventricular internal systolic diameter (LVID:S), ejection fraction and fractional shortening were measured. A) Representative echocardiography data from mice with netrin-1 (5 μg/kg) injected through the LV at the onset of reperfusion. B) Grouped data for left ventricular internal dimension (LVID) during systole (*p<0.05, **p<0.01 vs. I/R(vehicle)-1 day, ##p<0.01, ###p<0.001 vs. I/R(vehicle)-3 day) and diastole (*p<0.05 vs. I/R(vehicle)-1, # p<0.05 vs. I/R(vehicle)-3 day) for mice with netrin-1 injected through LV, n=3–7/group. C) Grouped ejection fraction and fractional shortening data for mice with netrin-1 injected through LV. *** p<0.001 vs. I/R (vehicle)-1 day, ###p<0.001 vs. I/R (vehicle)-3 day, n=3–7/group. D) Representative echocardiography data from mice with netrin-1 (5 μg/kg) injected through tail vein. E) Grouped LVID during systole (**p<0.01 vs. I/R(vehicle)-1 day, ##p<0.01, ###p<0.001 vs. I/R(vehicle)-3 day) and diastole (*p<0.05, **p<0.01 vs. I/R(vehicle)-1 day, #p<0.05, ##p<0.01 vs. I/R(vehicle)-3 day) from mice with netrin-1 (5 μg/kg) injected through tail vein, n=3–9/group. F) Grouped ejection fraction (*p<0.05, **p<0.01, ***p<0.001 vs. I/R (vehicle) -1 day, ##p<0.01, ###p<0.001 vs. I/R (vehicle) -3 day) and fractional shortening (* p<0.05, ***p<0.001 vs. I/R (vehicle) -1 day, #p<0.05, ###p<0.001 vs. I/R (vehicle) -3 day) from mice with netrin-1 (5 μg/kg) injected through tail vein, n=3–9/group. Data are presented as Mean ± SEM.

Figure 2. Intraventricular and Intravenous Delivery of Netrin-1 Improves Cardiac Function after I/R Injury in vivo

Echocardiography was performed on wild type C57BL6 mice reperfused for 24 hrs or 72 hrs after a 30 min left coronary artery (LCA) ligation. Left ventricular internal diastole diameter (LVID:D), left ventricular internal systolic diameter (LVID:S), ejection fraction and fractional shortening were measured. A) Representative echocardiography data from mice with netrin-1 (5 μg/kg) injected through the LV at the onset of reperfusion. B) Grouped data for left ventricular internal dimension (LVID) during systole (*p<0.05, **p<0.01 vs. I/R(vehicle)-1 day, ##p<0.01, ###p<0.001 vs. I/R(vehicle)-3 day) and diastole (*p<0.05 vs. I/R(vehicle)-1, # p<0.05 vs. I/R(vehicle)-3 day) for mice with netrin-1 injected through LV, n=3–7/group. C) Grouped ejection fraction and fractional shortening data for mice with netrin-1 injected through LV. *** p<0.001 vs. I/R (vehicle)-1 day, ###p<0.001 vs. I/R (vehicle)-3 day, n=3–7/group. D) Representative echocardiography data from mice with netrin-1 (5 μg/kg) injected through tail vein. E) Grouped LVID during systole (**p<0.01 vs. I/R(vehicle)-1 day, ##p<0.01, ###p<0.001 vs. I/R(vehicle)-3 day) and diastole (*p<0.05, **p<0.01 vs. I/R(vehicle)-1 day, #p<0.05, ##p<0.01 vs. I/R(vehicle)-3 day) from mice with netrin-1 (5 μg/kg) injected through tail vein, n=3–9/group. F) Grouped ejection fraction (*p<0.05, **p<0.01, ***p<0.001 vs. I/R (vehicle) -1 day, ##p<0.01, ###p<0.001 vs. I/R (vehicle) -3 day) and fractional shortening (* p<0.05, ***p<0.001 vs. I/R (vehicle) -1 day, #p<0.05, ###p<0.001 vs. I/R (vehicle) -3 day) from mice with netrin-1 (5 μg/kg) injected through tail vein, n=3–9/group. Data are presented as Mean ± SEM.

Figure 3. Netrin-1-mediated cardioprotection in vivo is DCC dependent

Myocardial infarction was induced by a 30 min left coronary artery (LCA) ligation followed by a 24 hr of reperfusion in DCC_+/+ mice and DCC_+/− mice. The mice were injected of 5 μg/kg netrin-1 at the onset of reperfusion. Netrin-1 yielded significant protection in DCC_+/+ and not in DCC_+/− animals. A) Representative TTC-stained heart slices from DCC_+/+, I/R+Netrin-1 group, and DCC_+/−, I/R+Netrin-1 group. B) Infarct size analyzed by percentage of area at risk divided by left ventricle (AAR/LV), infarct size divided by area at risk (Inf/AAR), and infarct size divided by left ventricle (Inf/LV). The results are represented as Mean±SEM. ***p<0.001 vs. DCC_+/+, I/R+netrin-1, n=7/group. I/R+UO126 I/R+Netrin-1

Figure 3. Netrin-1-mediated cardioprotection in vivo is DCC dependent

Myocardial infarction was induced by a 30 min left coronary artery (LCA) ligation followed by a 24 hr of reperfusion in DCC_+/+ mice and DCC_+/− mice. The mice were injected of 5 μg/kg netrin-1 at the onset of reperfusion. Netrin-1 yielded significant protection in DCC_+/+ and not in DCC_+/− animals. A) Representative TTC-stained heart slices from DCC_+/+, I/R+Netrin-1 group, and DCC_+/−, I/R+Netrin-1 group. B) Infarct size analyzed by percentage of area at risk divided by left ventricle (AAR/LV), infarct size divided by area at risk (Inf/AAR), and infarct size divided by left ventricle (Inf/LV). The results are represented as Mean±SEM. ***p<0.001 vs. DCC_+/+, I/R+netrin-1, n=7/group. I/R+UO126 I/R+Netrin-1

Figure 4. Netrin-1 induced cardioprotection in vivo is ERK 1/2 and NO dependent

Myocardial infarction was induced by a 30 min LCA ligation followed by a 24 hr of reperfusion. Mice were injected with either 5 μg/kg netrin-1; 200 μg/kg UO126 alone; 1 mg/kg PTIO alone; 5 μg/kg netrin-1/200 μg/k UO126; or 5 μg/kg netrin-1/1 mg/kg PTIO at the onset of reperfusion. UO126 was used to inhibit MERK1/2/ERK1/2 activity and PTIO was used to chelate NO specifically. Cardioprotection elicited by netrin-1 was suppressed in presence of UO126 or PTIO. Infarct size analyses as in Figure 2. The results were represented as Mean±SEM. ***p<0.001 vs. all others, n=7/group.

Figure 4. Netrin-1 induced cardioprotection in vivo is ERK 1/2 and NO dependent

Myocardial infarction was induced by a 30 min LCA ligation followed by a 24 hr of reperfusion. Mice were injected with either 5 μg/kg netrin-1; 200 μg/kg UO126 alone; 1 mg/kg PTIO alone; 5 μg/kg netrin-1/200 μg/k UO126; or 5 μg/kg netrin-1/1 mg/kg PTIO at the onset of reperfusion. UO126 was used to inhibit MERK1/2/ERK1/2 activity and PTIO was used to chelate NO specifically. Cardioprotection elicited by netrin-1 was suppressed in presence of UO126 or PTIO. Infarct size analyses as in Figure 2. The results were represented as Mean±SEM. ***p<0.001 vs. all others, n=7/group.

Figure 5. Netrin-1 activates ERK1/2 and eNOS/NO in vivo in a DCC dependent manner, while iNOS expression is unaffected

Shown are representative western blots and grouped densitometric data from DCC_+/+ and DCC_+/− mice treated at the onset of reperfusion with vehicle (normal saline), or 5 μg/kg of netrin-1. A) Grouped densitometric data of ERK1/2 phosphorylation that is normalized by ERK1/2. ***p<0.001 vs. corresponding DCC_+/+ mice, n=3/group. B) Grouped densitometric data of eNOS_s1177 phosphorylation that is normalized by eNOS. ***p<0.001 vs. DCC_+/+ mice, n=3/group. C) Representative and grouped data of iNOS expression, n=3/group.

Figure 5. Netrin-1 activates ERK1/2 and eNOS/NO in vivo in a DCC dependent manner, while iNOS expression is unaffected

Shown are representative western blots and grouped densitometric data from DCC_+/+ and DCC_+/− mice treated at the onset of reperfusion with vehicle (normal saline), or 5 μg/kg of netrin-1. A) Grouped densitometric data of ERK1/2 phosphorylation that is normalized by ERK1/2. ***p<0.001 vs. corresponding DCC_+/+ mice, n=3/group. B) Grouped densitometric data of eNOS_s1177 phosphorylation that is normalized by eNOS. ***p<0.001 vs. DCC_+/+ mice, n=3/group. C) Representative and grouped data of iNOS expression, n=3/group.

Figure 6. Netrin-1 induction of NO production in vivo is DCC and ERK 1/2 dependent

Bioavailable NO levels in the left ventricle were determined by electron spin resonance (ESR) from mice treated with either vehicle (normal saline), netrin-1 or netrin-1/UO126 at the onset of reperfusion (at same doses described earlier). Left panels show representative ESR spectra of NO production, while the grouped data are presented on the right. A) Netrin-1 markedly restores NO production which was reduced by I/R injury, and this response was inhibited by cotreatment with UO126. ***p<0.001 vs. Sham, ### p<0.001 vs. I/R+Netrin-1, n=5/group. B) Netrin-1’s effects on restoring NO production was abolished in DCC_+/− animals. ***p<0.001 vs. DCC_+/+, n=5/group.

Figure 7. Netrin-1 attenuation of mitochondrial superoxide production in vivo is DCC and ERK 1/2 dependent

Mitochondrial superoxide production was measured using electron spin resonance (ESR) using freshly isolated mitochondria from left ventricle after I/R surgery. A) I/R induced increase in mitochondrial superoxide production was attenuate by netrin-1 infusion, and this response was reversed by co-treatment of ERK1/2 inhibitor U0126. ***p<0.001 vs. Sham, ### p<0.001 vs. I/R+Netrin-1, n=5/group. B) Netrin-1’s effects on attenuating mitochondrial superoxide production was absent in DCC_+/− animals. ***p<0.001 vs. DCC+/+, n=5/group

Figure 8. Netrin-1 preservation of mitochondrial integrity during I/R in vivo is DCC and ERK 1/2 dependent

Calcium induced mitochondrial swelling was measured from freshly isolated mitochondria after I/R injury. A) Hearts were injected with vehicle, netrin-1 (5 μg/kg) or netrin-1+UO126 (200 μg/kg), at the onset of reperfusion. The data show that netrin-1 markedly reduced mitochondrial swelling, while co-treatment with UO126 abolished this protective effect. ***p<0.001 vs. Sham, ### p<0.001 vs. I/R+Netrin-1, n=5/group. B) Same experiments performed on DCC_+/+ and DCC_+/− animals. Data show that netrin-1’s protective effect against mitochondrial swelling was absent in DCC_+/− animals. ***p<0.001 vs. DCC_+/+, n=5/group.

Figure 9. Netrin-1 downregulates NOX4 expression and activity in vivo during I/R

Shown are representative western blots and grouped densitometric data from mice subjected to sham surgery, permanent LAD ligation for myocardial infarction (MI), or MI surgery with netrin-1 perfusion in osmotic minipumps. A) Western blots showing NOX4 expression. B) Grouped densitometric data of NOX4 protein expression that is normalized by Actin. Results are means ± S.E.M. # p<0.05 vs. I/R+Netrin-1, n=5/group. C) Grouped data shows that Fulvene-5 (a specific inhibitor of NOX4) sensitive NOX activity was increased with I/R, and netrin-1 perfusion eliminated this increase. Co-treatment with PTIO, a NO scavenger, abolished this effect of netrin-1. *p<0.05 vs. Sham, #p<0.05 vs. I/R+Netrin-1, n=4/group.

Figure 10

Netrin-1 attenuates autophagy in post-MI remodeled heart. Representative western blots and grouped densitometric data in mice subjected to sham surgery, permanent LAD ligation to induce myocardial infarction (MI), or MI surgery with netrin-1 infused using osmotic minipumps. A) Representative western blots of LCI/LCII and β-actin expression. B) Grouped densitometric data of LCII/LCI ratio after normalized by β-actin. Results are presented as Means±SEM. *p<0.05 vs. Sham, n=3/group.